The invention relates to methods for purifying polypeptide copolymers, especially COP-1 polypeptides.
Polymerization of natural or synthetic monomers is well known and described in early textbooks such as for example Organic Chemistry by Morrison and Boyd, 3rd edition, 1980, chapter 32. Polypeptides are a specific class of polymers formed by polymerization of amino acids.
“Copolymer polypeptides” are generally formed by a random co-polymerization of amino acids. In such co-polymerization, several different amino acids having the carboxy and/or amino groups activated are mixed together under reactive conditions, whereby the peptide link formation between the various amino acids present in the reaction mixture is random. It is also known in the art that reactive chemical groups that are not part of the polymer backbone, and should remain unaltered after polymerization, should be protected by protective groups. After the polymerization is complete these protective groups are chemically removed.
A method of forming a copolymer containing leucine and phenylalanine by random polymerization is described in U.S. Pat. No. 2,657,972.
U.S. Pat. No. 3,849,550 describes a method of forming a copolymer containing alanine (A), glutamic acid (E), lysine (K), and tyrosine (Y) by random polymerization. The method uses protected side groups, followed by two deprotection steps, but does not specify any purification steps. The resulting copolymer, which has subsequently been referred to as Copolymer-1 (COP-1) (see, e.g., U.S. Pat. No. 5,800,808), was reported as having a molar ratio of A:E:K:Y of 6:2:4.5:1.
U.S. Pat. No. 5,800,808 describes methods of obtaining COP-1 having a specific molecular weight profile, with subsequent purification by dialysis or ultrafiltration. The COP-1 was reported as having a molar ratio of A:E:K:Y of 6:2:5:1. The patent does not mention industrially applicable purification methods.
WO 2006/083608 describes methods of making COP-1 and mentions ultrafiltration to remove protecting groups and low molecular weight impurities. The molar ratio in the COP-1 of A:E:K:Y corresponds to ˜4.49:˜1.48:˜3.56:1. In an embodiment (Example 3), ultrafiltration was performed using a 5 kilodalton membrane to remove low-molecular weight impurities. After 6 cycles of ultrafiltration, the solution was acidified with acetic acid until a pH of 4.0 was achieved. Water was added and the solution was ultrafiltered until a pH of 5.5 was attained. The solution was concentrated and lyophilized for 60 hours.
US 2008/0021192 describes methods of making COP-1 and subsequently purifying the COP-1, such as by dialysis, chromatography, filtration, etc. The COP-1 reported has a molar ratio of A:E:K:Y corresponding to ˜4.49:˜1.48:˜3.56:1. In an embodiment (scheme 5), COP-1 is purified using ultrafiltration. A reaction mixture containing the COP-1 was filtered to remove any fine insoluble materials and the filtrate was passed through an ultrafiltration using a 1 kilodalton membrane first with circulating water until a pH of 8 was observed in the permeate and then circulating with 0.3% acetic acid in water to pH 5.5-6.0 in the retentate. The solution was then lyophilized to apparently obtain COP-1 as an acetic acid salt in solid form.
The prior art COP-1 ultrafiltration purification methods described above are multistep procedures suitable for lab scale experimentation. There remains a need, however, for an easier and/or more industrially applicable ultrafiltration method for purifying complex reaction mixtures comprising polypeptides, especially COP-1.